Daniel:Notebook/ComboLock/2017-3-24

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Linear Product Production[edit]

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This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.

However, as mentioned yesterday, first I want to make a linear product for an effective negative control. After reviewing the recommended protocol from NEB, it looks like I should use approximately 1 pmol of product per unit (uL) of enzyme I add. Yesterday, I added 4 uL of 100 uM circular product. For those of you who can do math: that's 400 pmol. Might have been a little too much. Modified protocol below will account for this.

Protocol[edit]

  1. Dephosphorylation (Padlock Oligo)
    1. Mix two of the the following reaction(s)
      1. 1 uL Padlock oligo (10 uM)
      2. 4 uL CutSmart Buffer (10X)
      3. 4 uL rSAP enzyme
      4. 31 uL nfH2O
    2. Incubate at 37C for 1 hour
    3. Incubate at 65 for 5 minutes
  2. Ligation
    1. Make reaction mixture according to table; Do not add ligase yet
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 Ligase Buffer 5X 1X 4
      Dephosphorylated Circ Latch (Step 1) 250 nM 1 pmol *This is less than the others 4
      Padlock 0601 oligo 10 uM 10 pmol 1
      C4 Positive Control oligo 10 uM 10 pmol 1
      C2 positive Control oligo 10 uM 10 pmol 1
      T4 DNA Ligase 5 U/uL 5 U 1
      nfH2O NA NA 8
      Total     20
    3. Use following thermocycler program
      1. Heat mixture to 95C for 5 min
      2. Lower temp by 0.2C per second to 55C
      3. Hold mixture at 55C for 15 minutes
      4. Lower temp to 20C by reducing 0.2C per second
      5. Hold at 20C
    4. Add 1 uL T4 DNA ligase
    5. Incubate at 20C for 30 minutes
    6. Heat kill enzyme by incubating at 65C for 15 minutes
    7. Add 20 uL nfH2O


    Continued tomorrow